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Trabajo presentado en el International Lipid Signaling and Oxidative Stress in Pathology Symposium, celebrado en Barcelona (España), los días 1 y 2 de diciembre de 2022, Niemann-Pick disease type C (NPC) is a slowly progressive lysosomal disorder whose principal manifestations are age dependent. The manifestations in the perinatal period and infancy are predominantly visceral, with hepatosplenomegaly, jaundice, and (in some instances) pulmonary infiltrates. From late infancy onward, the presentation is dominated by neurologic manifestations. The youngest children may present with hypotonia and developmental delay, with the subsequent emergence of ataxia, dysarthria, dysphagia, and, in some individuals, epileptic seizures, dystonia, and gelastic cataplexy. Although cognitive impairment may be subtle at first, it eventually becomes apparent that affected individuals have a progressive dementia

Trabajo presentado en el The Liver Meeting, celebrado en Washington, D.C. (Estados Unidos), del 4 al 8 de noviembre de 2022, Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease due to its association with obesity and type II diabetes. The presence of fibrosis indicates disease progression towards cirrhosis and hepatocellular carcinoma (HCC). Trafficking of cholesterol to mitochondria through increased expression of steroidogenic acute regulatory protein 1 (STARD1) has been described as a key factor for NAFLD progression. If this increase of STARD1 is cause or consequence, is still unknown. To examine this question we used a nutritional model of NASH with translational relevance to human disease with the aim to investigate the contribution of STARD1 in the progression of NASH in FRGN mice with humanized liver

Trabajo presentado en el The Liver Meeting, celebrado en Washington, D.C. (Estados Unidos), del 4 al 8 de noviembre de 2022, Systemic hypoxia is associated with an increase in cholesterol levels, which confers at higher risk for coronary heart disease. In addition, increased hepatic cholesterol has emerged as a important player in nonalcoholic fatty liver disease promoting the transition from steatosis to nonalcoholic steatohepatitis (NASH). As steroidogenic acute regulatory protein (STARD1) has been recently shown to promote NASH progression towards hepatocellular carcinoma (HCC) and its expression is regulated byhypoxia, our aim was to investigate the interplay between hypoxia signaling and the nutritional intake of cholesterol in the progression of NASH in mice with hepatocyte-specific deletion of von Hippel-Lindau (VHL) protein, an E3 ubiquitin ligase required for HIF degradation.

Trabajo presentado en el The Extracellular Matrix Pharmacology Congress, celebrado en Copenhagen (Dinamarca), del 23 al 25 de junio de 2022

Resumen del trabajo presentado en el 47º Congreso AEEH (Asociación Española para el Estudio del Hígado), celebrado en Madrid (España), del 9 al 11 de marzo de 2022, Background and Aims: Liver fibrosis is caused by an excessive accumulation of extracellular matrix (ECM) proteins. Macrophages are important effectors for ECM remodelling through phagocytosis and processing of the ECM within acidic compartments and can contribute to liver fibrosis resolution. Proteases, such as cathepsins, are essential for lysosomal proteolytic activity however, their contribution to ECM remodelling within the macrophages is unknown. Thus, the aim of this study was to investigate the proteolytic and degradative signalling pathways associated to macrophages during liver fibrosis. Method: To study the proteolytic and degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D. We generated and validated a novel macrophage-CtsD knock-out mouse strain by breeding LysMCre (macrophages) with CtsD floxed mice. Fibrosis was established by bile duct ligation at 7 and 14 days in CtsDF/F or CtsDΔMac mice and determined by hydroxyproline, α-SMA, Col1A1 and TGF-β RT-PCR in total liver. Collagen degradation and endocytosis was studied using DQ™ Collagen, type I and 10KDa Dextran probes, respectively and WB for Endo180 and UPAR in peritoneal macrophages. Lysosomal colocalization was demonstrated using LAMP2. Protease secretome was determined using Protease Array from R&D. Results: First, CtsD deletion in macrophages from CtsDΔ Mac mouse was confirmed by CtsD WB and gene expression in macrophages and dual IHP (F4/80-CtsD) in liver tissue. Of note, cathepsin B expression remained unaffected despite deletion of CtsD. CtsD deletion in macrophages, increased liver fibrosis after BDL as shown by an increase in liver hydroxyproline, and hepatic α-SMA, Col1A1 and TGF-β gene expression. No differences of liver damage were detected between CtsDF/F and CtsDΔMac after BDL. As expected CtsD activity increased only in CtsDF/F mice after BDL. Isolated CtsDΔ Mac macrophages showed a significant decrease in DQ™ Collagen, type I degradation versus CtsDF/F ones. Collagen degradative profile colocalized partially with LAMP2, indicating that collagen was degraded within the lysosome. Furthermore, Dextran endocytosis and Endo180 and UPAR collagen internalization receptors remained unaffected in both CtsDF/F and CtsDΔ Mac macrophages. Protease array in cell culture medium from CtsDF/F and CtsDΔ Ma c macrophages revealed CtsA, MMP7, MMP8 and MMP13 as part of CtsD proteolytic secretome. Conclusion: Lysosomal cathepsin D is essential for a correct collagenolytic activity displayed by macrophages during liver fibrosis and its absence contributes to the development of cholestatic-induced liver fibrosis

The authors < would like to correct the affiliations C and D that were not up to date, as follows: C: Research Unit on BioActive Molecules, Department of Biological Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), 08034, Barcelona, Spain and CIBEREHD, Barcelona, Spain D: Department of Biomedicina, Unitat de Biologia Cel.lular, Centre de Recerca Biomèdica CELLEX, Institut d′Investigacions Biomédicas August Pi i Sunyer (IDIBAPS), Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036, Barcelona, Spain> The authors would like to apologise for any inconvenience caused.

Trabajo presentado en las XV Jornadas CIBER Enfermedades Hepáticas y Digestivas, celebradas los días 8 y 9 de noviembre del 2021 en formato híbrido (presencial-virtual) en Barcelona (España), Changes in proteolytic activity are essential to liver fibrosis development. Proteases control not only matrix turnover but also, the activation and repression of growth factors and chemokines influencing disease progression. However, our knowledge about the proteolytic enzymes contributing to liver fibrosis is still very limited. Thus, the aim of this study was to analyse CtsD cell-specific role during liver fibrosis.

Trabajo presentado en las XV Jornadas CIBER Enfermedades Hepáticas y Digestivas, celebradas los días 8 y 9 de noviembre del 2021 en formato híbrido (presencial-virtual) en Barcelona (España), Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease due to its association with obesity and type II diabetes. NAFLD encompasses a spectrum of disorders ranging from steatosis to nonalcoholic steatohepatitis (NASH), an intermediate stage of fatty liver disease which is characterized by steatosis, hepatocellular death, fibrosis, oxidative stress and inflammation. The presence of fibrosis indicates disease progression towards cirrhosis and hepatocellular carcinoma (HCC). Aim: On the base of the liver-humanized FRGN KO model, we set out to establish an in vivo NASH model using a NASH-induced Trafficking of cholesterol to mitochondria through steroidogenic acute regulatory protein 1 (STARD1), which has been described as a key factor for NAFLD progression. If the increase of STARD1 is cause or consequence of NASH progression is still unknown. Although significant advances in NASH pathogenesis have been achieved using experimental models, the translational impact of these treatments to patients has been disappointing, reflecting the need to improve experimental animal models and an accurate understanding for NASH pathogenesis. Using this humanized mouse model with NASH, we describe the contribution of STARD1 overexpression in the progression of the pathogenesis.

Trabajo presentado en el International Workshop on Liver and Gut Fibrosis, celebrado en Valencia (España), los días 7 y 8 de octubre de 2021, Background and Aims: Changes in proteolytic activity are essential to liver fibrosis development. Previous reports suggest that macrophages are important effectors for ECM remodelling through phagocytosis and processing of ECM within acidic compartments. However, the role of macrophages in ECM remodelling during liver fibrosis is unknown. Methods: To study the degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D (CtsD). We generated and validated a novel macrophage-CtsD knock-out mouse by breeding LysMCre (macrophages) with CtsD floxed mice. Fibrosis was established chronically by CCl4 (0.5μl/g) or bile duct ligation in CtsDF/F or CtsDΔMac mice and determined by SR staining, hydroxyproline and fibrogenic genes by RT-PCR. Collagen degradation and endocytosis was studied using DQ™ Collagen type I and 10KDa Dextran probes respectively in peritoneal macrophages. Lysosomal colocalization was determined by IF using LAMP2. WB for Endo180 and UPAR and dual IF (Collagen- F4/80) was performed in fibrotic livers. Results: First, CtsD deletion in macrophages from CtsDΔMac mouse was confirmed by WB and RT PCR in macrophages and dual IF (F4/80-CtsD) in liver tissue. Of note, cathepsin B expression remained unaffected despite deletion of CtsD. CtsDΔMac mice demonstrated enhanced liver fibrosis in both CCl4 and BDL models as shown by an increase in Sirius red staining, hydroxyproline and hepatic Col1A1 and TGF-β mRNA. CtsDΔMac macrophages showed a significant decrease in DQ™ Collagen type I degradation versus CtsDF/F ones. DQTM collagen profile partially colocalized within the lysosomes (LAMP2). No affectation of the endocytosis process in macrophages or the expression of the collagen receptors UPAR and Endo180 was detected in livers from CtsDF/F or CtsDΔMac after CCl4 challenge. Finally, F4/80+ Collagen loaded macrophages were detected in CCl4 livers. Conclusions: Lysosomal cathepsin D is essential for the correct collagenolytic activity displayed by macrophages during liver fibrosis.

Resumen del trabajo presentado en el International Liver Congress, celebrado de manera virtual del 23 al 26 de junio de 2021, Background and aims: Changes in proteolytic activity are essential to liver fibrosis development. Previous reports suggest that macrophages are important effectors for extracellular matrix (ECM) remodelling via its phagocytosis and processing within acidic compartments. However, the role of macrophages in ECM remodelling during liver fibrosis is unknown. Thus, the aim of this study was to investigate the proteolytic and degradative pathways associated to macrophages during liver fibrosis. Method: To study the degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D (CtsD). We generated and validated a novel macrophageCtsD knock-out mouse by breeding LysMCre (macrophages) with CtsD floxed mice. Peritoneal macrophage polarization from CtsDΔMyel+/+ or CtsDΔMyel−/− mice was achieved with LPS for M1 or IL4/IL13 for M2 stimulation. Polarization markers and MMP profile were assessed by RT-PCR. Collagen degradation and endocytosis was studied using DQ™ Collagen, type I and 10 kDa Dextran probes respectively, and WB for Endo180 and UPAR. Lysosomal colocalization was determined using LAMP2. Fibrosis was established chronically by CCl4 (0.5μl/g) in CtsDΔMyel+/+ or CtsDΔMyel−/− mice and determined by SR staining, α-SMA IHP and Col1 WB as well as RT-PCR. Results: First, CtsD deletion in macrophages from CtsDΔMyel−/− mouse was confirmed by WB, CtsD activity in macrophages and dual IHP (F4/80-CtsD) in liver tissue. M1 and M2 polarization of CtsDΔMyel+/+ and CtsDΔMyel−/− macrophages using LPS (10 and 50 ng/ml) or IL4/ IL13 (20 and 50 ng/ml) resulted in similar and significant induction of iNOS (M1) and CD206 (M2) gene expression respectively. However, while M1 downstream effector CCl2 was similarly induced between CtsDΔMyel+/+ and CtsDΔMyel−/− macrophages after LPS stimulation, IL10 was defectively induced in M2-primed CtsDΔMyel−/− macrophages. In addition, both, M1 and M2 CtsDΔMyel−/− macrophages displayed defective induction of MMP-2, -3 and -7 gene expression. CtsDΔMyel−/− macrophages showed a significant decrease in DQ™ Collagen, type I degradation versus CtsDΔMyel+/+ macrophages at 37C. As expected, no degradation was detected at 4C. Collagen degradative profile colocalized partially with LAMP2, indicating that collagen was degraded within the lysosome. Furthermore, Dextran endocytosis in both CtsDΔMyel+/+ and CtsDΔMyel−/− macrophages remained unaffected. CtsDΔMyel−/− mice presented enhanced liver fibrosis as shown by an increase in Sirius red staining, alpha-SMA and Col1A1 gene and protein expression with no affectation of the Endo180/UPAR collagen internalization receptors (WB). Conclusion: Lysosomal cathepsin D is essential for a correct collagenolytic activity displayed by macrophages during liver fibrosis.

Additional file 1: Table S1. General ICP-MS parametersused for Zn analysis.

Trabajo presentado en el Extracellular Matrix Pharmacology Symposium, celebrado digitalmente los días 24 y 25 de marzo de 2021, Background: Changes in proteolytic activity are essential to liver fibrosis development. During fibrosis there is a dysregulation between extracellular matrix (ECM) synthesis and degradation, resulting in excessive ECM accumulation. Previous reports suggest that macrophages are important effectors for ECM remodelling through phagocytosis and processing of ECM within acidic compartments. However, the role of macrophages in ECM remodelling during liver fibrosis is unknown. Objectives: Thus, the aim of this study was to investigate the proteolytic and degradative pathways associated to macrophages during liver fibrosis. Methods: To study the proteolytic and degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D. Accordingly, we generated and validated a novel macrophage-CtsD knock-out mouse strain by breeding LysMCre (macrophages) with CtsD floxed mice. Peritoneal macrophage polarization from CtsDΔMyel+/+ or CtsDΔMyel-/- mice was achieved with LPS for M1 or IL4/IL13 for M2 stimulation. Polarization markers and MMP profile were assessed by real-time-PCR. Collagen degradation and endocytosis was studied using DQ™ Collagen, type I and 10KDa Dextran probes respectively, and WB for Endo180 and UPAR. Lysosomal colocalization was determined using LAMP2. Finally, fibrosis was established chronically by CCl4 (0.5μl/g) in CtsDΔMyel+/+ or CtsDΔMyel-/- mice and determined by SR staining, α-SMA IHP and Col1 WB as well as RT-PCR in total liver. Results: First, CtsD deletion in macrophages from CtsDΔMyel-/- mouse was confirmed by WB, CtsD activity in macrophages and dual IHP (F4/80-CtsD) in liver tissue. To note, cathepsin B expression remained unaffected despite deletion of CtsD. M1 and M2 polarization of CtsDΔMyel+/+ and CtsDΔMyel-/- macrophages using LPS (10 and 50 ng/mL) or IL4/IL13 (20 and 50 ng/mL) resulted in similar and significant induction of iNOS (M1) and CD206 (M2) gene expression respectively. However, while M1 downstream effector CCl2 was similarly induced between CtsDΔMyel+/+ and CtsDΔMyel-/- macrophages after LPS stimulation, IL10 was defectively induced in M2-primed CtsDΔMyel-/- macrophages. In addition, both, M1 and M2 CtsDΔMyel-/- macrophages displayed defective induction of MMP-2, -3 and -7 gene expression. CtsDΔMyel-/- macrophages showed a significant decrease in DQ™ Collagen, type I degradation versus CtsDΔMyel+/+ macrophages at 37C. As expected, no degradation was detected at 4C. Collagen degradative profile colocalized partially with LAMP2, indicating that collagen was degraded within the lysosome. Furthermore, Dextran endocytosis in both CtsDΔMyel+/+ and CtsDΔMyel-/- macrophages remained unaffected. CtsDΔMyel-/- demonstrated enhanced liver fibrosis as shown by an increase in Sirius red staining, alpha-SMA and Col1A1 gene and protein expression with no affectation of the Endo180/UPAR collagen internalization receptors (WB). Conclusion: Lysosomal cathepsin D is essential for a correct collagenolytic activity displayed by macrophages during liver fibrosis.

Trabajo presentado en las Jornadas CIBEREHD, celebradas online, del 9 al 11 de noviembre de 2020, Changes in proteolytic activity are essential to liver fibrosis development. Proteases control not only matrix turnover but also, the activation and repression of growth factors and chemokines influencing disease progression. Beyond metalloproteases, our knowledge of the proteolytic enzymes contributing to liver fibrosis is limited. The importance of lysosomal protease cathepsin D (CtsD) in kidney fibrosis has been recently demonstrated but its role in liver fibrosis remains elusive. Thus, the aim of this study was to analyse CtsD cells-specific role during liver fibrosis.

Trabajo presentado en las XIII Jornadas Científicas CIBEREHD, celebradas en Barcelona (España), del 25 al 26 de noviembre de 2019, Among emergent metabolic chronic liver diseases, nonalcoholic fatty liver disease (NAFLD) and its more advanced form, nonalcoholic steatohepatitis (NASH), are becoming a major public health problem in industrialized countries. NAFLD is the most common liver disease, affecting 20¿40% of adults. 10-30% of patients with NAFLD have NASH, and the accompanying fibrosis may progress to cirrhosis and hepatocellular carcinoma (HCC). HCC is one of the most fatal and fastest-growing cancers. Recently, non-alcoholic steatohepatitis (NASH) has been recognized as a major HCC promoter. Animal models are an essential tool for the identification of the mechanisms driving the pathogenesis and progression of NAFLD to NASH and HCC. Ideally, experimental models should reflect the etiology, disease progression, and pathology of human NAFLD. Unfortunately, currently available models, MCD diet, Western diet, and high-fat diet, are complementary and each of them partially reflects the real picture of human NAFLD.

Trabajo presentado en el Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD) / Liver Meeting, celebrado en San Francisco del 9 al 13 de noviembre de 2018, [Background] Cholesterol accumulation in mitochondrial membranes is a hallmark of alcoholic and non-alcoholic fatty liver diseases and hepatocarcinoma. In physiological conditions mitochondrial cholesterol levels are low, however in pathological conditions cholesterol accumulates within this organelle contributing to the progression of the disease. Cholesterol changes membrane physical properties and impacts negatively in the action of specific solute carriers (e.g. Scl25a11) but its role on mitochondrial function and organization of respiratory chain components has not been deciphered yet. Thus, our aim was to study the effects of cholesterol in the function, morphology and respiration of mitochondria from mice liver., [Methods] WT C57BL/6J male mice were fed for 2 days with normal chow (Ctrl) or highcholesterol diet (2% Cholesterol and 0,5% Sodium Cholate, HC). A wide range of mitochondrial analysis were subsequently performed to determine: free cholesterol levels by HPLC and immunofluorescence; Membrane fluidity by fluorescence anisotropy; Morphology by Electron Transmission Microscopy; Dynamics by Western Blot; Respirometry by Seahorse XFe 24 Analyzer; and Respiratory supercomplexes assembly by BlueNative Page., [ Results] HC feeding for two days resulted in a significant increase in the mitochondrial cholesterol levels (2-3 fold), which distributed in both the outer and inner membranes, accompanied by increased fluorescence anisotropy indicative of reduced membrane fluidity. This outcome correlated with increased liver damage assessed by serum ALT levels. Interestingly, mitochondrial cholesterol accumulation resulted in morphological changes of mitochondria that appeared swollen, rounded and with disrupted cristae. In addition, cholesterol decreased expression of mitochondrial fission inducer p-DRP1. High-resolution respirometry analyses using pyruvate+malate revealed that cholesterol loading caused a decreased mitochondrial respiratory profile. Both Respiratory Control Ratio (RCR) (state 3/state 4o respiration) and Uncoupling Control Ratio (UCR) (State 3u/ State 4o respiration) werediminished in mitochondria loaded with cholesterol. In consonance with these observations, a decrease in the respiratory chain complexes assembly into the functional quaternary structures called supercomplexes (III2 +IV, I1 +III2 and I1 +III2 +IV) was observed in cholesterolenriched mitochondria. Conclusion: These data indicate that the accumulation of cholesterol in mitochondrial membranes not only disrupt membrane physical properties but also alters mitochondrial morphology and dynamics, and the functional organization of respiratory supercomplexes assembly.

Trabajo presentado en el Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD) / Liver Meeting, celebrado en San Francisco del 9 al 13 de noviembre de 2018, [Background] Valproic acid (VPA), an anticonvulsant drug and acetaminophen (APAP), a pain reliever, are among the most prescribed drugs worldwide. We have previously shown that VPA pretreatment sensitized to APAP hepatotoxicity in non-fasted mice. VPA targets mitochondria, induces mitochondrial permeability transition and alters glutathione (GSH) homeostasis. APAP can cause liver necrosis and its major mechanism of hepatotoxicity is the generation of NAPQI, which depletes GSH and covalently bind to proteins. Although, both drugs cause endoplasmic reticulum (ER) stress, the mechanisms and impact of VPA plus APAPinduced ER stress remains poorly understood. Therefore, we tested whether GSH depletion could mediate VPA plus APAPinduced ER stress and the impact of N-acetylcysteine (NAC) or tauroursodeoxycholic acid (TUDCA) treatment in liver injury and GSH homeostasis., [Methods] Non-fasted WT mice were treated three times every 12h with VPA (400mg/Kg, sc) followed by a single dose of APAP (300 mg/Kg, ip) and 1 hour after the APAP injection, we administered a single dose of NAC or TUDCA (2,5 mmol/kg). Liver damageby ALT and H&E, TUNEL and GSH homeostasis was examined. Expression of ER stress markers were examined by qPCR and WB. Primary mouse hepatocytes (PMH) were treated with Tunicamycin (10µg/ml), diethylmaleate (DEM) (0.8mM), VPA (0.5mM), APAP (15mM) and NAC (20mM) to examine GSH levels and ER stress markers., [Results] VPA plus APAP caused acute liver failure revealed by the H&E, TUNEL staining and the serum transaminases levels, that was preceded by the induction of NAPQI-protein adducts, GSH depletion (total and mitochondrial pools) and ER stress markers (CHOP, Ire-1a) and the expression of StARD1. TUDCA or NAC markedly protected against VPA plus APAP-induced liver damage and ER stress induction and StARD1 upregulation. Moreover, NAC but not TUDCA prevented VPA plus APAP total GSH depletion but both restored mitochondrial GSH levels. PMH treated with DEM reduced total GSH levels and induced expression of CHOP. Conclusion: The results indicate that GSH regulates ER stress response and that GSH depletion by VPA plus APAP causes ER stress, which in turn contributes to the hepatotoxicity induced by synergism of both drugs in non-fasted mice.

Trabajo presentado en el Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD) / Liver Meeting, celebrado en San Francisco del 9 al 13 de noviembre de 2018, [Background] Alcoholic liver disease (ALD) is a spectrum of disorders that begin with hepatic steatosis, which can progress to alcoholic steatohepatitis (ASH) and cirrhosis. The incomplete understanding of mechanisms contributing to ALD progression has limited the availability of effective therapy. Mitochondrial dysfunction is a hallmark of ALD. Altered mitochondrial morphology in human ALD and indirect metabolic determinations suggested impaired mitochondrial function in ALD patients. In experimental models, impaired mitochondrial respiration seems to be species-dependent (rats vs. mice), with evidence in mice showing a correlation between increased mitochondrial respiration and ALD severity. Therefore, we examined the mitochondrial function from alcohol-fed FRG mice xenotransplanted with human adult hepatocytes (HH)., [Methods] FRG mice were injected with HH through the spleen followed by controlled cycles of NTBC withdrawal. Humanized FRG mice were fed an alcohol (5%, ethanol; 36% calories) or control liquid diets for 10 days after 5 days of acclimation. HH were isolated and oxygen consumption rates (OCR) determined by a flux analyzer to examine respiratory parameters. In parallel, hepatocyte mitochondrial respiration was determined in wild type mice fed alcohol diet for comparison. In some cases, perivenous (PV) and periportal (PP) hepatocytes were isolated from wild type mice fed alcohol., [Results] FRG mice were repopulated with HH by 80-85%, as estimated by the determination of serum human albumin levels and expression of fumaroylacetoacetate by immunohistochemistry. OCR significantly increased in HH from FRG mice fed alcohol compared to FRG mice fed control diet. This outcome translated in a significant increase (2-fold) in basal OCR, ATP production and maximal respiration, without changes in proton leak, coupling efficiency or mitochondrial mass (cytochrome c immunostaining). Interestingly, these findings in OCR and respiratory parameters were similar to those found in mitochondrial respiration from wild type mice fed alcohol for the same period of time. Moreover, the mitochondrial respiratory alterations reflected predominant changes in PV rather than PP hepatocytes from wild type mice fed alcohol. Conclusion: Using this subrogate model of humanized ALD, our findings show that alcohol feeding increases mitochondrial respiration in human hepatocytes metabolizing alcohol, suggesting that increased mitochondrial respiration may contribute to human ALD.